WW . w Mill/Ill!l!!!lllllllllllllllllllllfl 29310063 W I ”LIBRARY 9’2 Michigan State University This is to certify that the thesis entitled STUDIES ON THE ECOLOGY 0F NEMATODES ASSOCIATED WITH VEGETABLES GROWN IN ORGANIC SOILS presented by James Kotcon has been accepted towards fulfillment of the requirements for M. S. degreeinBOtany and Plant Pathology @flv/g/ ‘ Major professor Date August '2, 1979 0-7 639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. STUDIES ON THE ECOLOGY OF NEMATODES ASSOCIATED WITH VEGETABLES GROWN IN ORGANIC SOILS By James Kotcon A THESIS Submitted to Michigan State university in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1979 ABSTRACT STUDIES ON THE ECOLOGY OF NEMATODES ASSOCIATED WITH VEGETABLES GROWN IN ORGANIC SOILS By James Kotcon Population dynamics of three plant-parasitic nematodes, four pre- daceous nematodes and endomycorrhizal fungi were evaluated in association with carrots, celery, onions and weed hosts to determine interactions of biotic and abiotic factors in organic soil agro-ecosystems. Objec- tives included quantifying extraction and sampling procedures, monitor- ing environmental factors and evaluation of host-symbiont interactions. Population density changes of plant-parasitic nematodes were asso- ciated with host crop; however, horizontal and vertical distributions were not influenced. Population densities of predaceous nematodes were influenced by the associated crop, but did not influence the pOpulation density of plant-parasitic nematodes. Some weed species were good hosts for Meloidogyne hapla and Pratylenchus penetrans; however, population densities of Paratylenchus hamatus declined. Controlled environment studies demonstrated improved growth of onions infected with the endomycorrhizal fungus, Glomus fasciculatus, decreased growth in some cultivars when infected with y. hapla, and protection against nematode damage when jointly inoculated. To John Davenport whose technical advice and long hours of patient assistance made much of this work possible. ACKNOWLEDGEMENTS I wish to express a most sincere appreciation to my major professor, Dr. G. W. Bird, for his assistance and guidance during this research, and for his critical evaluation of this manuscript. I would also like to thank the members of my advisory committee for their advice and assistance, Dr. John Lockwood, Dr. Peter Murphy, and Dr. Gary Simmons. Thanks also to Mrs. Natalie Knobloch, Ms. Lindy Meitz and Mr. Charles Benner for their technical assistance; to my fellow graduate students, Alma Elliot, Joe Noling, and Bob VanArkel for many helpful discussions; to the Department of Entomology for the use of research facilities; and to Ms. Laura Meal for many hours of data analysis and typing. Finally, a special thanks to all of my friends and comrades for their support and encouragement over the course of this work. TABLE OF CONTENTS LIST OF TABLES O O O I O O O O O O O O O O 0 O O O O O 0 LIST OF FIGURES O O O O O O O O 0 O O O O O I O 0 O O O O INTRO DUCTI ON 0 O O O O O O O O O O O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . Extraction Procedures . . . . . . . . . . . . . . . . Sampling Techniques . . . . . . . . . . . . . . . . . . Physical Factors Influencing Nematode Distribution . , Biotic Factors Influencing Nematode Distribution . . . Role of Host and Symbionts . . . . . . . . . . . . . . EVALUATION OF EXTRACTION PROCEDURES . . . . . . . . . . . Methods 0 O O 0 O O O O O O O O O O O O O O O O I O O 0 Comparison of Extraction Procedures . . . . Quantification of the Centrifugal-Flotation Method ReSUJ-ts O O O O O O O O O O O O O I O O O O O O O O O I Comparison of Extraction Procedures . . . . . Quantification of the Centrifugal-Flotation Method SWLING TECHNIQUES . O O O O O O O O O O O O O O O O O O MethOdS O O O O O O O O O O O O O O O O O O O O O O O 0 Results . . . . . . . . . . . . . . . . . . . . . . . . Horizontal Distribution . . . . . . . . . . . . . Changes in Dispersion . . . . . . . . . . . . . . Vertical Distribution . . . . . . . . . . . . . . PHYSI CAL FACTORS O O O O O O O O O O O O O O O O O O O O O MethOdS O O O O O O O O O O O O O O O O O O O O O O O 0 Results 0 O O O O O O 9 O O O O O O O O O O O O O O O 0 8011 Temperature 0 O O O O O O O O O O O O I O O 0 8011 Nutrient Status 0 O O O O O O O O O O O O O 0 iv V1 viii \ONWW 12 16 26 26 26 27 28 28 3O 32 33 35 35 38 40 4O 4O 40 41 TABLE OF CONTENTS (continued) BIOTIC FACTORS INFLUENCING PLANT-PARASITIC NEMATODES Me thOdS O O O O O O O O O O O O O O O O O O I O 0 Results 0 C O O O O C O O O O O O O O O O O O O 0 Influence of Weeds on Plant-Parasitic and Predaceous NematOdeS O O O O O O O O O O C O O U 0 Population Dynamics of Predaceous Nematodes Interactions of Concomitant Nematodes . . . Influence of Chemical Nematicides on Nematodes and Mycorrhizae Associated With Onions . INFLUENCE OF THE HOST AND SYMBIONTS . . . . . . . . Me thOdS I O O O O O O O I O O O C O O O O O O O 0 Effect of Host Crop on Population Densit of Plant- Parasitic Nematodes . . . . . . . . . . Effect of Inoculum Density of VAM on Onion . Effect of Inoculum Density of M} hapla on Onion Response of Onion Cultivars to M, hapla . . VAMFNematode Interactions with Onion . . . . Results . . . . . . . . . . . . . . . . . . . . . Effect of Host Crop on Population Density of Parasitic Nematodes . . . . . . . . . . Host Responses to Nematode Infection . . . . Effect of Host Crop on VAM Spore Density . . Effect of VAM Inoculum Density on Onion . . Effect of M. hapla Inoculum Density on Onion Response of Onion Cultivars to M. hapla . . Joint Action of VAM and Nematodes on Onions DISCUSSION . . . . . . . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . . . APPENDIX A: Root Staining Techniques . . . . . . . APPENDIX B: Nematode Distribution Maps . . . . . . APPENDIX C: Nematode Dispersion Indices . . . . . . LITERATIJRE CITED 0 O O O O O O O O O O O O O O O O O 46 46 47 47 52 56 59 63 63 63 64 64 65 65 66 66 69 72 72 72 76 79 86 93 95 98 108 115 1.1 1.2 2.1 2.2 3.1 3.2 4.1 4.2 4.3 4.4 5.1 5.2 5.3 LIST OF TABLES Average population densities extracted from field soils by three techniques . . . . . . . . . . . . . . Extraction efficiency of the centrifugal-flotation technique for six nematode species . . . . . . . . . Nematode frequency distributions from four subplots and the whole plot sampled November 1, 1977 . . . . . Vertical distribution of plant-parasitic nematodes at the MSU MUCk Farm 0 I O O O O O O O O O O O O O 0 Degree day accumulations on 1978 sampling dates at three soil depths calculated using the Baskerville- Emin method at a 10 C base . . . . . . . . . . . . . Pearson correlation coefficients of soil test results with various biotic parameters in plot C-l7 of the MS U Mu Ck Farm 0 O O O O O I O O O O O O O C O O O O Nematode pOpulation densities and VAM spore densities associated with nematicide treatments in onion production . . . . . . . . . . . . . . . . . . . . . Weed hosts of plant-parasitic nematodes . . . . . . . Pearson correlation matrix of initial pOpulation den- sities of seven nematode species and vesicular-arbus— cular mycorrhiza (VAM) spores . . . . . . . . . . . . Pearson correlation matrix of population densities of seven nematode species and vesicular-arbuscular mycorrhiza (VAM) spores at harvest . . . . . . . . . Influence of plant density on the yield of three vegetable crops . . . . . . . . . . . . . . . . . . . Influence of Glomus fasciculatus on the growth and VAM infection of onion (cv Downing Yellow Globe) after Sixteen weeks 0 O O O O O O O O C O O O O C O O O C 0 Influence of Meloidggyne hapla on the growth of onions (cv Downing Yellow Globe) . . . . . . . . . . . . . . . vi 29 31 36 39 42 45 48 60 60 61 71 74 75 LIST OF TABLES (continued) 5.4 5.5 5.6 5.7 5.8 5.9 Influence of Meloidogyne hapla on the growth and development of five cultivars of onions . . . . . . Influence of five onion cultivars on the population density of Meloidogyne hapla . . . . . . . . . . . Joint action of Glomus fasciculatus and Meloidogyne hapla on onion (cv Downing Yellow Globe) growth parameters over seven weeks . . . . . . . . . . . . Joint action of Glomus fasciculatus and Meloidogyne hapla on onion (cv Downing Yellow Globe) growth parameters over fifteen weeks . . . . . . . . . . . Joint action of Glomus fasciculatus and Meloidogyne hapla population density parameters on onion para- meters on onion (cv Downing Yellow Globe) over seven weeks 0 O O O O O O O C O O O O O O O O O C I O O I Joint action of Glomus fasciculatus and Meloidogyne hapla population density parameters on onion (cv Downing Yellow Globe) over fifteen weeks . . . . . vii 77 78 8O 81 83 84 3I1 4.1 4.2 4.3 4.4 4.5 4.6 SIl 5.2 5.3 5.4 5.5 LIST OF FIGURES Cumulative degree days at three soil depths during 1978 I I I I I I I I I I I I I I I I I I I I I I I I I Plant-parasitic nematodes infesting weed subplots . . Predaceous nematodes associated with weed subplots . . Influence of associated crop on population dynamics of Mbnonchoides spp. . . . . . . . . . . . . . . . . . Influence of associated crop on population density of MylonChUIUS SEEI I I I I I I I I I I I I I I I I I I I Influences of associated crop on population density of MononChuS $22 I I I I I I I I I I I I I I I I I I I I I Influence of associated crop on population dynamics of Ajorcelaimus SEE I I I I I I I I I I I I I I I I I I I Influence of host crop on population density of P, Penetrans I I I I I I I I I I I I I I I I I I I I I I Influence of host crop on population density of M. 118213 I I I I I I I I I I I I I I I I I I I I I I I I Influence of host crop on population density of P. hamatus I I I I I I I I I I I I I I I I I I I I I I I Influence of host crop on Spore density of g. faSCiCUIatus I I I I I I I I I I I I I I I I I I I I I Effect of G. fasciculatus and M. hapla on onion growth viii 43 51 53 54 55 57 58 67 68 7O 73 82 INTRODUCTION Vegetable production is an important aspect of Michigan agriculture with a large portion being produced on organic soils. Unfortunately, many of these crops are highly susceptible to attack by plant-parasitic nematodes. Annual losses to Michigan agriculture due to nematodes is estimated at approximately $80,000,000. An important tool for control of nematode losses became available with the discovery of the nematicidal properties of ethylene dibromide and the 1,3-dichloropropenes in the 1940's. By 1970 an estimated $51 million worth of nematicides were sold in the U.S., most of which were applied to high value crops (such as fruits and vegetables), often with dramatic yield increases (119). The broad spectrum of biological activity of most nematicides, especially fumigant nematicides, has been known for some time (2, 24); however, due to the increasing envir— onmental awareness of the last decade, an increased interest in the ecological effects of nematode control practices has appeared. Increases in crop yield have been attributed not only to reductions in plant-parasitic nematode populations, but also to changes in soil microbe populations (2, 57, 88, 89, 124) including increased mycorrhizal infection (11), and interference with nitrogen transformations in soil resulting in increased nitrogen available to the plant for growth (24, 93). The effect on nontarget, beneficial organisms such as mycorrhizal fungi (11, 89) or nematode—predaceous fungi (64, 65) are of critical importance. Current knowledge regarding these and other interactions is insufficient for viable management decisions and more information is needed in order to maximize crop yields while reducing environmental dis— ruptions. The objectives of this research are to study the interactions of biotic and abiotic factors influencing the population ecology of three important nematodes in Michigan vegetable production, Meloidogyne hapla Chitwood 1949, Pratylenchus penetrans (Cobb 1917) Sher and Allen 1953, and Paratylenchus hamatus Thorne and Allen 1950. Complete coverage of all ecological factors affecting these nematodes is beyond the scope of this thesis; however, a number of biological factors which have previously received insufficient attention in Michigan were studied, including ef- fects of vesicular-arbuscular mycorrhizae, predaceous nematodes, weeds and host crop influences. The broad spectrum of this research necessarily resulted in a diffuse collection of data which will be integrated into this thesis. LITERATURE REVIEW Extraction Procedures Before realistic estimates of nematode population dynamics can be made, methods must be standardized and evaluated for their efficiency in extracting nematodes from soil and root tissue. A number of techniques have been proposed, each with advantages and disadvantages. One of the earliest described methods is the Baermann Funnel. Christie and Perry (20) described a modification of this technique which, in various forms, is still used today. The basic principle involves movement of the nema- tode from a soil sample through a cheese cloth or filter paper lining after which they swim or settle to the bottom of the sealed neck of the funnel. After several days, the sample is drawn off and counted. Var- ious modifications have been proposed (1, 69, 116) but all depend on movement of the nematode, are inefficient in collection of some taxa (116), and are subject to errors resulting from egg hatch during the extraction period (63). In 1955, Caveness and Jensen (16) described a centrifugal-flotation method. This technique, as modified (54) is one of the most important methods in use today. The soil sample is suspended in water, then sieved and centrifuged to concentrate the nematodes in the centrifuge pellet. A sucrose solution (or salt solution, eg. MgSO4 (69)) of spe- cific gravity 1.1 to 1.25 (usually 1.13) is used to resuspend the nematodes. A second centrifugation removes most soil particles while leaving the nenatodes in the supernatant which is sieved. Nematodes are washed off the sieve for observation or counting. The efficiency of this technique varies with a number of soil characteristics and proced- ural modifications (62). The technique is fast, reliable and suitable for extraction of all nematodes (living and dead), a number of fungal spores and a variety of soil invertebrates. Many other procedures exist. Probably the most accurate method was described by Minderman (69). The soil sample is stained and placed in a counting dish for direct observation. The process, although precise, can only be used with small samples and is extremely tedious. Elaborate extraction procedures and modifications are described by Seinhorst (101, 102). An elutriator is used to separate nematodes from soil by an up— ward current of water which washes the nematodes through a series of sieves for collection. The process is modified by various sieving pro- cedures and by changing the flow from 350 to 700 cm/hr. An upward flow of 250 to 350 cm/hr collected 86-89% of the Ditylenchus _p, added to a soil sample. Higher flow rates were needed to extract larger nematodes such as Dorylaimus sp,, Mononchus s2, and Criconemoides s2. This method has since undergone further modifications and semi-automatic elutriators for nematode extraction are being produced commercially (97). This method has the advantage of being able to accomodate large soil samples (500 to 1500 cm3). Some soil types, however, can still give problems and extraction efficiency varies with nematode species and body length. The main source of error in the centrifugation-flotation method and the elutriation method is in sieving the nematodes from large volumes of water. If sieve openings are less than one-tenth the length of the nematode, a reasonably high efficiency of recovery can be obtained. Using a 50 mm sieve, almost all Hoplolaimus _p, (1.5 mm), 75% of Dit - lenchus s2, (0.8 mm) and 65% of Pratylenchus s2, adults (0.6 mm) were recovered when sieving through 4 liters of water. However, only 25% of Pratylenchus s2, juveniles (0.3 mm) were recovered and numerous re- peated sievings were needed to retain a high percentage. Larger vol— umes of water pulled more nematodes through the screen with correspond- ingly reduced extraction efficiency (101). Although the above techniques are widely used, information on their efficiency is sparse. Most com- parisons have been concerned with the relative efficiency of the tech— niques used and are highly empirical (63, 81, 82, 112). Quantitative data for extraction efficiency of the total soil population are reported by Seinhorst (101, 102) and by Minderman (69). As mentioned, the elu- triation method can give highly variable results under varying condi— tions. Minderman's modification of the Baermann pan technique with alternate shaking and settling gave 96% recovery in 18 hours as com- pared to direct counts of stained controls. Flotation methods are re- ported to give 85-90% of soil nematodes but the proportion lost in sieving was not specified (81). Other methods are described by Murphy (74). Quantification of endoparasitic nematodes presents problems in that a large fraction of the population may be feeding inside roots and is not recovered by standard soil extraction methods. Migratory endopar- asites can be recovered by soaking the roots in water as in a Baermann pan for several days. A mist chamber is also commonly used. These methods may require several weeks to extract a high proportion of nema- todes (8). Several solutions are known to enhance migration of 25351: lenchus _p, from roots. When incubated on a gyratory shaker, a mixture of ethylethoxy mercuric chloride and dihydrostreptomycin sulfate (EMC- DSS) at 10 and 50 ppm was found to extract 90% of the recoverable P, brachyurus within 96 hours whereas more than two weeks were required to obtain a similar percentage using water (8). Unfortunately the total number in roots at the beginning of the experiment was not determined and so the actual extraction efficiency was not determined. Barker and Nusbaum (5) suggest that only about 50% of the nematodes are re- moved by such methods in 2 weeks. They were able to obtain a second similar yield by enzymatic maceration combined with sieving and the Baermann Funnel. Sedentary endoparasites (e.g. Meloidogyne spp.) lose mobility with- in several days of penetrating the root and therefore cannot be extracted by the shaker-EMC-DSS method. Second-stage larvae which are removed are most likely to be the result of egg hatch and are a poor indication of the actual level of infestation. Staining of a root sample is re- quired to obtain accurate counts. Several techniques are discussed in Appendix A (48, 66, 84). Another technique, described by Dropkin et a1 (27) involves a maceration of roots with pectolytic enzymes. Large numbers of live adult Meloidogyne §P° females were recovered from galled roots. A rapid technique for estimating the number of eggs in soil has also been developed (15). Its use, however, is limited to egg masses such as those of Meliodogyne spp. Roots and egg masses are sieved from soil with water, the gelatinous matrix is dissolved in NaOCl, and eggs are stained in acid fuchsin in 25% lactic acid to facilitate counting. Centrifugal—flotation is probably best suited for determining numbers of single eggs in soil as would be produced by most ectoparasitic nema- todes (32). Since these methods are seldom used, an unknown proportion of a nematode population is unaccounted for in most surveys. It should be noted, however, that eggs from most nematodes are very difficult to identify to a specific taxon. Quantification of this segment of a ne- matode population is virtually impossible in the forseeable future. Sampling Techniques Although significant advances have been made in nematode extraction techniques, population estimates are only as good as the sample from which they were obtained. The distribution of nematodes in the field is variable, both horizontally and vertically and single samples are inappropriate unless a known uniform distribution exists (10). Statis- tical analyses of nematode distribution under field conditions are rarely found in the literature. Nematodes are usually considered to be "randomly" distributed in most field trials although an aggregated or clumped distribution is probably more common. When a non-random distribution is found, it is often correlated with some factor such as soil type (5), vegetation (131), previous cropping history (126) etc. Analysis of frequency data may indicate whether the population conforms most closely to a uniform, random, or aggregated distribution which can be approximated statistically by a normal, Poisson, or negative binomial frequency distribution (123). This kind of information may give a more fundamental basis for determining the number of samples which are needed to adequately determine the population density of a given field. In most cases, a number of subsamples should be combined and a composite sample analyzed. The number of subsamples necessary varies with each situation (58). More samples are needed for a five acre field than for sampling a single fruit tree (9). This is due to the large differences in populations of plant-parasitic nematodes which can occur within short distances of each other (5, 126). Several basic sampling techniques are available. Goodell and Ferris (42) were able to make detailed comparisons of several methods and patterns of sampling using a computer analysis of data from a field plot. From this they found that several uniform or random patterns gave similar results and suggested that a minimum of 10-12 subsamples be composited for each sample (personal communication). In fields with greater variability more subsamples may be appropriate. An alternative sampling method, referred to as sequential sampling, is gaining p0pu- larity. The basic rationale is to simply determine if a population is above or below a given threshold level. Samples are taken and evalu— ated sequentially until enough evidence has accumulated to make a judge- ment, within a specific confidence level, as to whether a population is above or below the threshold level. At the extremes of very high or very low populations, only a few samples are needed to make such a judgement; whereas, if moderate densities are found, more samples are taken until the population can be placed at one level or the other. Under many circumstances, this method can result in substantial savings in time, cost, and energy. It is less effective at quantifying the actual population level as it is designed to determine qualitatively if the population is above or below a given density (80). Physical Factors Influencing Nematode Distribution The most obvious factor in nematode distribution is soil type and its characteristics. The physical make-up of soil particles varies from heavy clays to silt to coarse and all possible mixtures. The relative proportion of these particles and the structure of soil aggregates determine the size and number of pore spaces in which nematodes live. Movement of nematodes is impossible if pore spaces are too small. Nema- todes live in a water lens at the juncture of soil particles and must cross from one lens to another across the surface of the soil aggregates when moving. If the aggregates are too large or the soil is dry, the water surface tension may be sufficient to inhibit movement (122). The optimum size for mobility is when the ratio of nematode length to parti- cle diamter is about 3:1 (113). Soil moisture influences nematode movement and survival. High soil moisture content results in reduced oxygen, higher CO2 levels, and lower osmotic and matric water potentials. Wet soils absorb heat more slowly, resulting in smaller temperature gradients and lower tempera- tures, whereas dry soils subject the nematode to a more extreme environ- ment. Reduced temperature and lower oxygen tension slow nematode move- ment and reduce infection (122, 128). As a general rule, the Optimum for P, penetrans activity is at field capacity (56) although this re- lationship becomes less obvious as the amount of fine silt and clay increases. 10 The effects of temperature on M, hapl§_are fairly well defined, with respect to the major segments of the life cycle. At 0 C, 41% of Mh‘hapl§_eggs survived and were infective after 90 days and larvae survived for 16 days. Larvae were infective for up to 12 months as temperatures increased to 10 C but declined rapidly at warmer tempera- tures (113). Eggs will survive short periods of freezing temperatures (-30 C or colder) if given an acclimation period at near-freezing temp- eratures. Larvae do not survive freezing below -5 C in pure water. The formation of ice crystals apparently disrupts internal tissues. Salt solutions, however, greatly increase larval survival at -7 C (95). Egg hatch occurs rapidly at 18, 21, and 27 C, with over 25% of the eggs hatching in 12 days. Hatch is slower, however, at 12 C and at 36 C. The optimum temperature is about 21 C, although the rate of hatch changes considerably with incubation time and with the age of the egg masses (129). These data appear to support a cumulative heat units concept (degree days) as controlling egg hatch. This hypothesis may be involved in other time-dependent activities, e.g., reproduction, growth, survival, etc., although other factors such as high temperatures will complicate the issue. Root invasion by M. hapla occurs at temperatures as low as 5 C is optimum from 15 to 20 C and does not occur above 30 C. Growth and reproduction will occur above 15 C with the optimum from 20-25 C (113). Thomason and Lear reported reduced reproduction above 30 C and complete failure above 33.6 C (114). Slightly warmer temper- atures (21-26 C) were needed for optimum movement and root penetration in muck soils (128). These and other differences may be attributed to 11 differences in water relations between organic and mineral soils, and therefore results cannot readily be extrapolated from one soil type to the other (24). Soil pH has little direct effect on nematode populations. It exerts its primary effect on plant growth and nutrient availability in soil solution. Population differences due to pH can be attributable to changes in host vigor and growth and not in activity to nematodes (122). Variation in soil chemistry can cause major changes in nematode populations. Compounds affecting nematodes originate from a number of sources including microorganisms (especially bacteria), root exudates, decaying organic matter, chemicals added by man (fertilizer, pesticides, etc.) and secretions from nematodes. A number of ions, amino-acids, monosaccharides, and a variety of toxic breakdown products affect nema- tode behavior (122). Decomposing nitrogenous compounds release NH3 which is toxic to nematodes (121). Organic amendments have been used with limited success to control populations. The direct toxic effects are difficult to assess due to concomitant increases in nematode-trap- ping fungi and other antagonistic organisms. Root exudates are important in attracting nematodes to plants. Work by Klinger (56) showed that both exogenously applied CO2 and heat sources may be attractive to nematodes. These types of interaction with soil and root compounds deserve considerably more attention in order to provide a better understanding of nematode behavior and popu- lation dynamics (96). An expanded discussion of the influence of soil chemistry can be found in recent books by Norton (77) and Wallace (123). 12 Biotic Factors Influencing Nematode Distribution Biological factors affecting populations of plant-parasitic nema- todes can be divided into two groups: (1) those which influence nema- todes in the root, and (2) those which influence free-living stages in soil. The general role of a host and its symbionts are of overriding importance and are treated separately. Biological factors that have a marked effect on nematode populations include predation, parasitism, disease, competition, toxin secretion and secretion of other compounds which disrupt normal nematode behavior and development. Several re- views of biological nematode controls have been published and give a good discussion of these biotic factors (19, 28, 96, 125). Several viruses, bacteria, and sporozoans cause diseases of nema— todes, and can kill 25 to 50% of the plant-parasitic nematodes in in- fected populations. Diseases of eggs, free-living larvae and adult forms are known (96, 125, 127). Natural control by predaceous and endoparasitic fungi was discussed in an early review by Duddington (28) and was the subject of a recent book by Barron (6). Endoparasitic fungi infect nematodes by (l) in— gested spores which lodge in the buccal cavity, (2) adhesive spores which adhere to the cuticle of passing nematodes, and (3) flagellate spores which can track a host via exudate gradients from nematode ori- fices and will encyst on the cuticle. The spores germinate and infect the nematode through openings or directly through the cuticle. Hyphae ramify throughout the host tissue resulting in mortality. At maturity hyphae usually grow out of the host and produce more spores. l3 Predaceous fungi form adhesive hyphae which hold the nematode while it is being infected, or a net or ring structure to trap and hold the nematode. One of the more interesting occurrences in hematology is the constricting ring of some predaceous Deuteromycetes. These consist of three-celled rings which, when stimulated by a nematode passing through, rapidly swell inward thus pinching the nematode body and holding it until infection occurs (6). There is currently some debate regarding the feeding status of predaceous fungi. Webster concluded that nematode- trapping fungi were independent of nematode populations, were often able to live saprophytically on soil organic matter, and required the release of certain soluble carbohydrates to stimulate trap formation (125). Barron, however, found no convincing evidence for a saprophytic potential in predatory fungi and suggested nematodes were their sole food source (6). It is generally concluded that many of the endopara- sites are relatively specific to nematodes. The status of predaceous fungi needs more research and probably will vary among species (96). The effect of soil fumigants on predaceous fungi has been investi- gated (63, 65). Ethylene dibromide and 1, 2-dibromo-3—chloropropene are fungistatic to some predaceous fungi. Growth, however, can resume after the fumigant is removed or degraded. Other fumigants (1, 3 dich- loropropene, 1,3-D + methyl isothiocyanate and sodium methyl dithiocar- bamate) are fungitoxic and probably result in extensive damage to pre- datory fungal populations. Predaceous nematodes have potential for biological control of plant-parasitic nematodes (19, 125). Thorne (115) studied populations of Mononchs relative to Heterodera schachtii. He found that the highest l4 predator population production was in spring. They became quiescent during the summer months. He also reported infection by a sporozoan in 30% of the predator population and suggested this reduced predator numbers. Rhabditis s22, were a preferred food source for Mononchs, possibly because of their softer cuticle. He concluded that although the highest population production was in spring and coincided with hatching of H, schachtii, the reduced activity during summer months in- dicated that Mononchs were not important in controlling H, schachtii. This was confirmed by Linford and Oliveira (61) who suggested that certain Dorylaimoid nematodes (Dorylaimus, Discolaimus and Actinolaimus Spp.) were more important as predators than Mononchus spp. In one study M} pappilatus was the only predator of four species which pre- ferred nematodes to other soil organisms (19). A recent study on Mononchoides potohikus (130) suggested that, al- though it could be cultured solely on bacteria, its predation rate on Mesorhabditis littoralis was unaffected by the presence of bacteria. This nematode apparently has no selectivity for prey species (although cannibalism was observed only once) and the rate of predation is deter- mined strictly by the number of prey it encounters. Feeding was con- tinuous as the nematodes apparently were never satiated. Feeding by Seinura tenuicaudata was selective. It fed on Aphelenchus avenae, M. hapla, P. penetrans, Neotylenchus linfordi, Ditylenchus s2. and second stage larvae of Heterodera trifolii, but not on Xiphinema $22., Hoplolaimus galeatus, or adult females and cysts of H. trifolii. Cannibalism was observed only in starving populations. It continued until only a few remained. This nematode feeds by puncturing the 15 cuticle of prey with the stylet and secreting a compound which apparently kills or paralyzes the prey instantly. The body contents are then re- moved through the stylet, leaving the empty cuticle (47). The variability in prey preference is of great importance in the use of predaceous nematodes for biological control. In most cases, soils which are heavily amended with organic matter will experience a dramatic, although temporary, increase in predaceous nematode population, as well as predaceous fungi and other soil predators. This usually results in only short term reductions in plant-parasitic populations. This, how- ever, may be sufficient to protect a crop during the most susceptible early stages of growth. Nematode predation has also been reported by a wide variety of other organisms including Collembola, mites, Tardigrades, Enchytraeids, Turbellarians and amoebae (26, 96, 125). The relative importance of these organisms is largely unknown. A better understanding of these factors is essential to successful management of plant-parasitic nema- todes. A factor which is often overlooked is the influence of weed hosts in nematode disease epidemiology. Weeds can exert a considerable com- petitive influence on crops (51, 120) and may be important in terms of allelopathic interactions (85). Weeds can serve as reservoirs of in- oculum for reinfection or overwintering (25, 29). They can also influ- ence soil microorganism communities and therefore indirectly alter the disease potential of plant-parasitic nematode populations. In most cases, fields will be infested with more than one plant- parasitic nematode species. If these infect the same plant, they can l6 enhance, reduce, or have negligible effect on the growth and develop- ment of the concomitant species. In most cases, ectoparasitic nematodes have less influence on dis- ease complexes than endoparasites (103, 105). Sikora et al (103) found that P. penetrans was unable to increase on bent grass unless Meloidogyne .BEEEE.W35 also present. M, Eééfil populations, however, were lower when it had to compete with P. penetrans or Tylenchorrhynchus agri. Using a split root technique on tomato, M, incognita was unaffected by P, penetrans on the opposite root half; however, P. penetrans popula— tions were lower and root penetration was reduced when the opposite root half was inoculated with M, incognita. Apparently P, penetrans simply competed for root sites to reduce M, incognita populations; how- ever, M, incognita induced a systemic influence to alter the relation- ship between P, penetrans and the host (30). In another study, Turner and Chapman (118) found that M, incognita had no effect on the number of P, penetrans entering alfalfa roots. .3. penetrans, however, reduced M, incognita invasions at high inoculum densities (118). These results were reconciled by Gay and Bird (36) in their discussion of a hypothesis on the interaction of concomitant populations. Populations will be en- hanced by another species if the host is resistant to the second species but are suppressed if the host is susceptiable to the second species. This interaction is complicated by the production of necrotic or hyper- plastic symptoms. Role of Host and Symbionts The discussion of biological factors which influence nematode pep- ulation dynamics must consider what is clearly the most important l7 component of a nematode ecosystem, the host and its symbionts. This subject is extremely complex as it involves a holistic approach. The variety of organisms that could interact with the host is nearly end- less and each is potentially able to alter nematode populations. An interesting example was described involving celery and three plant para- sitic nematodes (109, 110). Field soil was found to induce much greater root necrosis and yield loss than could be obtained from sterile soil inoculated with M, 22212, A wide variety of microorganisms was isolated from necrotic roots, but only Pythium polymorphon caused necrosis, with or without M, ha2la. ‘M, ha2la inoculations greatly increased Pythium infection of roots. When Pratylenchus penetrans was inoculated into field soil, it reduced celery growth but did not increase necrosis or Pythium infection. Root populations of this nematode were higher in field soil than in autoclaved soil. Paratylenchus projectus had no detrimental effects on Pythium interactions with celery (110). A second study showed that the increased necrosis was due to enhancement of Pythium infection by nematode galls (109). This obviously will affect the success of nematode reproduction and reinvasion of subsequent gener- ations. Many important symbiotic associations involve mycorrhizal fungi. This aspect of the hosts' development is described in detail to assess the variety of mechanisms by which such an interaction affects nematode population dynamics. Symbiotic associations of fungi with plant roots can be characterized into two types. Ectomycorrhizae are found pri— marily on tree roots (especially Gymnosperms) and contain a fungal mantle surrounding the host root as well as intercellular growth in the 18 cortex (referred to as the Hartig net). Endomycorrhizae are ubiquitous, being found in most vascular plants, and are comprised of a loose fungal network in soil which invades roots and grows inter- and intracellularly. Septate and nonseptate fungi have been described. The latter are re- ferred to as vescular-arbuscular mycorrhizae (VAM) because of their morphological structures produced internally in the root and are con- sidered to be the most agronomically important type. VAM exhibit a wide range of symbiotic characteristics, the impor— tance of which has only recently been realized and reviewed (38, 72, 73, 94). The ectomycorrhizal fungi have also been reviewed recently (106, 132). Mycorrhizal fungi infecting root tissue were first reported in the early 1880's (13). The early literature includes figures of vesicles and, later, arbuscules. The fungi were first classified as "Rhizophagus" but were later shown to be related to Endogone‘g22. The early work showed no pathogenic influences and the beneficial symbiotic role has been discussed for many years (46, 86). The beneficial influence of VAM has recently been demonstrated by numerous workers. The improved uptake of phosphorous as well as other mineral nutrients is well estab- lished and believed to be primarily responsible for the growth increases associated with mycorrhizal plants. The taxonomy of VAM fungi is extremely complex and needs revision. VAM are members of the family Endogonaceae. For many years they were described as species of the genus Endogone. This genus has recently been divided into four genera (39). Mycorrhizal forms are described from Gigaspora, Glomus, Acaulospora and Sclerogystis. The species used in most of the greenhouse experiments described in this thesis is l9 Glomus fasciculatus (sensu Gerdemann 1974) and is described in most of the earlier literature as Endogone fasciculata. Considerable confusion exists, especially in earlier literature, as to the influence of VAM on plant growth. This is primarily because different fungal species exert different influences on host relationships. Until the taxonomy is well defined, the precise influences will probably remain uncertain. The morphology of VAM is dependent on both the host and fungal species (37). In general, the fungus exists as spores in the soil. After germination, hyphae grow randomly until a suitable root is found. An appressorium is formed on the root surface and penetration occurs in epidermal cells or root hairs. The hyphae are irregular in sizes, shape and contortions. Coarse, thick-walled nonseptate hyphae ramify throughout the soil for a distance of several centimeters. Smaller, thin-walled spores and vesicles are formed in soil. Internally, the hyphae grow both inter- and intracellularly throughout the root cortex. Arbuscules are formed by repeated branching of intracellular hyphae, often filling a large proportion of the cell volume. These are usually short—lived structures. Within three days, the smallest branches appear to be redigested by the host until only the main trunk of the arbuscule remains. The host nucleus enlarges and becomes associated with the center of the arbuscule. The arbuscules continue to form near the ad— vancing edge of fungal infection. Although reinvasion (M5 host cells has been reported, most arbuscules are associated with the advancing edge of fungal infection and are usually only found near root tips. Much is still unknown regarding the interaction between host and fungal symbiont at the cellular level. 20 As hyphae ramify throughout root tissue and reach maturity, ovate to spherical vesicles are formed. These may be inter- or intracellular (37), are thin or thick-walled, and are believed to function in food storage or, possibly, in reproduction since they may resemble azygo- spores or chlamydospores. As the root matures, the primary cortex is sloughed off. The fungus maintains connections between soil and root hyphae as long as the cortex is intact (76). New infections of branch roots occur from soil hyphae. VAM tissue has not been observed in the endodermis, stele or the actively growing root tips and is lost com— pletely when the primary cortical tissues are lost. The physiological aspects of spore germination, infection, repro- duction, and other functions of the symbiotic relationship are largely unknown. The relationship is obligate for the fungus as it has never been successfully grown in culture. The spores will germinate in cul- ture, however, the mycelium dies when the food reserves of the spore are exhausted (38). The host plant, however, can grow and, with a high fertilization regime, achieve normal development under sterile conditions. Spore germination was studied by Mosse (70). She found high germ- ination on a soil agar plate but that germination was inhibited by soil or by autoclaved soil. She speculated that a substance produced by active soil organisms induces germination and found it to be water sol- uble, dialysable, and is counteracted by fungistatic principles in the soil. In the same study, Mosse (70) found that the hyphae were not attracted to aseptic roots, and infected roots only through chance encounters. Infection usually occured 5 to 10 days after inoculation and may require three or more weeks. Under aseptic conditions (71) 21 infection never occurred unless an unidentified Pseudomonas spp. was present in the media, or unless pectic enzymes were added. The active factor was suggested to be a bacterial compound produced under low ni- trogen nutrition (adding N as N03, NHZ, asparagine or urea completely inhibited VAM infections on agar media). Penetration stimulated mycelial growth in the soil (18). As the infection continues, starch is withdrawn from host tissues to provide the carbon source for hyphal growth (38). At the same time, the hyphae take up phosphate from soil and transport it to roots, some- times from a distance of up to 7 cm (37, 87). A soluble alkaline phos- phatase was identified as being specific to VAM associations (40). Other ions which are taken up may induce similar enzymes to facilitate transport to roots. The amount of growth stimulation is strictly dependent on the level of infection in the root. This is related to the number of spores in the soil. Although only one spore is needed to constitute an "effective" inoculum (73), in most cases, larger numbers of spores result in higher infection levels (22). The infection level is dependent on a number of other factors. Soil phosphorous in high concentrations is particularly effective in inhibiting growth and infection by the fungus. Menge et a1 (67) used a split root technique to show that root phosphorous con- centrations determined the level of infection and suggested that high root phosphorous concentrations could induce an incompatible physiologi- cal response to VAM regardless of the soil phosphorous levels. Other factors of importance in reducing VAM infection in roots include light intensity (34) and high nitrogen concentrations in soil. These 22 conditions have been correlated with reduced carbohydrate levels in the root, making the root a less favorable medium for growth (38). The degree of infection is also dependent on the age of roots and other factors (107). The competitive ability of some VAM plants is increased and results in improved growth especially when under competitive stress from other plants (44). The beneficial influence of VAM on growth has warranted field trials to test the effectiveness of introducing VAM species into agricultural systems. Field inoculations may be possible as a seed treatment (35) or after fumigation to remove competing fungi from soil (89). Unfortunately, only limited success was reported from these attempts. Ectomycorrhizal interactions result, in some cases, in reduced infection by disease causing organisms. These interactions were re- viewed by Zak (132). The influence of VAM on pathogenic organisms is not well known and has been described in only a few interactions (97). Most of the available literature is based on cotton, soybean or tobacco diseases. Thielaviopsis basicola infections can be reduced in mycorrhizal plants (3, 4, 100). The interactions of Pythium and Phytophthora root rots are more variable. Ross (92) found increased root rot by EBXEQ‘ 2hthora megaspermae var. sojae (PMS) race 1 on a susceptible soybean cultivar but not on a resistant cultivar. VAM, however, decreased kill from PMS race 3 in another study (18). Pythium root rot was unaffected by VAM and neither pathogen had any effect on VAM. Virus multiplication was stimulated by VAM or high phosphorus fertilization, but the effect 23 on mycorrhizae was not reported in this study (23). Meloidogyne incggf nita populations decreased on VAM tobacco (4), but increased under some soybeandmycorrhizae combinations (99). On tobacco, Heterodera solanac- earum and VAM reduced the reproductive capacity of each other and re- sulted in lower yields of both susceptible and resistant cultivars than with Heterodera or VAM alone (33). Meloidogyne and Heterodera were both associated with reduced field spore densities, however, Trichodorus, Pragylenchus and Heliocotylenchus had no effect on spore populations (98). On cotton, Pratylenchus brachyurus showed almost no effect on either VAM sporulation or plant growth although mycorrhizae resulted in significant growth increases in infected plants (53). Few conclusions can be drawn from these studies. Endoparasitic nematodes seem to have more influence on VAM than do ectoparasitic nematodes. Fungal patho- gens, however, show no clear pattern in VAM interaction studies. It is hoped that with more studies a more comprehensive theory of pathogen- mycorrhizae interactions will become apparent. The role and significance of a large preportion of soil fauna and flora concomitant with plant—parasitic nematodes is largely unknown. While direct interactions seem to be unlikely, the indirect influence of soil fungi, bacteria, actinomycetes, protozoa and other microbial organisms appear to be important in terms of competition for substrate, production of toxins and antibiotics, alteration of behavior through metabolite secretions, etc. Unfortunately, the tools for studying these interactions are underdeveloped and only a limited knowledge can be inferred from those studies which have been undertaken. 24 One tool which has been used is soil fumigation. An excellent series of papers was published in 1976 in which soil microbe populations were followed for up to one year following fumigation. Nitrifying bac- teria became reestablished rather quickly after methyl bromide fumiga- tion but were suppressed for at least 60 days by chloropicrin. This means that most of the nitrogen in the soil was available as NH4 and not N03 (93). Other soil bacteria populations fluctuated widely with rapid declines followed by increases 10 to 1000 times the normal level. The total population of soil flora antagonistic to Gaeummanomyces gram- igi§_increased although relative proportions of some flora declined (88). Soil fungi were able to survive although their populations were depressed and reinvasion was slow especially at lower depths (124). In a similar study, some fungal species were permanently eliminated by methyl bromide (96). Population dynamics studies of Olthof and Potter (78, 79) are im- portant in determining the role of plant-parasitic nematodes in crop yield. Their main objective was to determine economic thresholds for M, 222l§_and P, 2enetrans on a variety of vegetable crops. They reported that population levels of 2000.35 22212_per kg soil or 666 2, 2enetrans per kg soil were sufficient to reduce onion yields to the point where fumigation became economical. Other crops were studied and economic thresholds determined. The degree of susceptibility or tolerance of a host plant to a nematode is determined by a combination of genetic and environmental factors. It may involve characteristics such as attraction to roots (59) or a variety of other environmental parameters controlling nematode 25 growth and development. The overriding characteristic involves the genetic susceptibility of the host. Rohde (91) defined resistance as "a set of characteristics of the host plant which act more or less to the detriment of the parasite". This definition bases resistance on nematode survival and reproduction and not on host health. The primary basis for parasitism is nutritional. Resistance is usually due to the failure to supply an essential nutrient, but can also result from lack of response to the nematode e.g., syncytial formation (7), plant defense mechanisms such as chlorogenic acid hypersensitivity (91) or phenolic acid accumulation (104), or differences in response to environmental conditions (45). The general subject of population ecology of plant-parasitic ne- matodes has been reviewed by Norton (77) and Wallace (123). These books are recommended to the reader interested in the population dynam— ics and nematode ecology. EVALUATION OF EXTRACTION PROCEDURES The plant response to nematode infestations depends on the rela- tionship between the nematode population density and the food resources supplied by the plant (105). A major portion of this research is de— voted to the quantitative assessment of populations involved in disease situations. Methods Comparison of Extraction Procedures: A series of experiments was used to assess the efficacy of several procedures for extraction of nematodes from soil. The carrot bioassay, modified Baermann pan and centrifugal-flotation techniques were compared using soil samples collected from plot C-l7 of the Michigan State Uni- versity Organic Soils Research Farm (hereafter referred to as MSU "Muck Farm"). Plot C-l7 is 15.24 by 60.96 m and was previously (1977) used for a carrot nematicide trial. No statistically significant (P = 0.05) dif- ferences among treatments were found in the initial, mid-season or final population densities of M, 222l2, The plot was divided into 100 subplots (3.05 m2) and soil samples were taken November 4, 1977 by combining 8 to 10 cores (2.5 cm in diameter, 10 to 20 cm deep) from each subplot. Similarly, samples were taken from 25 sub-subplots (0.6 m2) subdivided from each of four randomly selected subplots. All soil samples were mixed thoroughly and a 100 cm3 aliquant was analysed for nematodes using each of the three methods. 26 27 Carrot bioassays were conducted by placing the soil sample in a 5.0 cm diameter plastic pot and planting pre-germinated carrot seeds (cv Gold Pak). Carrots were grown in the greenhouse for 5 to 8 weeks, after which the roots were washed, stained (Appendix B) and the number of M. Ma_2_l_a_ infecting roots counted. The modified Baermann pan consisted of a standard 9 inch pie pan with the bottom replaced by a coarse screen. Two Kleenex tissues were laid across the pan and the soil sample placed on the tissues. The pan was placed in an uncut pie pan with enough water to saturate the tissue and soil sample. After incubating for 48 hours at 25 C, the upper pan and soil sample were removed. The remaining water was poured onto a 400-mesh (38 um) sieve, the nematodes rinsed into an 18 x 200 mm test tube stored at 12 C until counted. Finally, a set of soil samples was processed using the centrifugal- flotation method as described by Jenkins (29). Due to the time limita- tions involved, this method was used almost exclusively for all further investigations and was therefore examined more intensively to determine absolute extraction efficiency. Quantification g: the Centrifugal-Flotation Method: A series of preliminary experiments was conducted by adding a known number of nematodes to an autoclaved soil sample and processing the sample. In this manner, a number of minor modifications in technique were derived which resulted in a method which consistently gave a rela- tively high extraction efficiency. The preliminary studies also iden- tified components of the procedures where the greatest losses occured. 28 Although a few nematodes remained sedimented in the pellet after the final centrifugation with sucrose, the largest losses occured when ne— matodes were poured onto sieves and slipped through the meshes. Nematodes were obtained from infested soil by the Baermann pan method as described except that the nematodes were concentrated by settling and decanting instead of sieving to prevent biased samples. Eight aliquants of motile stages of M. 23212.2, 3. 2enetrans, P. hamatus, Heliocotylenchus $22., Tylenchorrhynchus spp. and Mononchus 322. were counted and then poured into 100 cm3 samples of autoclaved muck soil. The soil samples were processed by the standardized centrifugal-flota- tion technique and the number of nematodes recovered was determined. The experiment was repeated once. The data were combined for each species and the extraction efficiency constant was determined. Results Comparison 2£_Extraction Procedures: The relative extraction efficiency of the three techniques varied with the nematode involved. The bioassay was effective only for M. .22212, Although vermiform nematodes were observed, the stain completely obscured all morphological features making taxonomic identification im- possible. The presence of M, Mg2l§ was detected by the host's response of galling and giant cell formation and by presence of swollen stages of the nematodes. There was no significant difference between the Baermann pan method and the centrifugal-flotation method when comparing the number of P, 2enetrans recovered. Significantly more 2, hamatus and M. 22212, how- ever, were obtained by centrifugal-flotation than by other methods (Table 1.1). 29 TABLE 1.1 Average population densities extracted from field soils by three techniques. Nematodes/100 cm3 Extraction Procedure M, ha2la .23 hamatus g, 2enetrans . 1 Carrot Bioassay 3.1 a - - Baermann Pan 3.5 a 1.8 a 3.8 a Centrifugal-Flotation 5.4 b 4.2 b 3.4 a lColumn means followed by the same letter are not significantly different (P = 0.05) by the Student-Newman-Keuls Multiple Range Test. 30 Quantification 2£_the Centrifugal-Flotation Method: Preliminary attempts to determine the actual extraction efficiency revealed considerable fluctuation in nematode recovery. Significant variability was found using different centrifuges, varying settling times, different sized sieves, etc. When these were standardized, the number of nematodes passing through a given sieve was determined. Using a 400 mesh sieve (38 um opening), 25 to 40% of M, 22213 larvae (mean length = 369 um) were recovered each time a sample was sieved in preliminary experiments. Greater than 75% of larger nematodes, such as g, 2enetrans or Criconemoides 522., were recovered from sieving. These types of data confirm those of Seinhorst (101) and showed that recovery from sieving was dependent on the length of the nematode and the size of the sieve openings. Extraction efficiency varied from 10% for Mononchus 222. to 58% for Helicotylenchus 822. (Table 1.2). The low values for Mononchus s22. were unexpected and may be a result of the low number of nematodes used or may reflect an intrinsic characteristic of these nematodes which restricts their extraction by these methods. The reciprocal of the fraction of nematodes extracted was used as the extraction efficiency coefficient in subsequent studies to adjust the data to give an estimate of the actual soil population of each nematode in each soil sample. 31 TABLE 1.2 Extraction efficiency of the centrifugal-flotation tech— nique for six nematode species. Nematode Initial Recovery Std. Dev. Extraction population (%) (%) coefficient M. M2213 1691 28.1 11.1 3.56 P, 2enetrans 731 36.7 7.5 2.72 P, hamatus 573 17.1 5.1 5.85 Heliocgtylenchus 222, 217 57.7 24.7 1.73 Tylenchorrhynchus s22, 65 37.4 34.3 2.67 Mononchus s22, 31 10.1 17.6 9.90 SAMPLING TECHNIQUES Wallace (123) describes nematode distributions as random, uniform or patchy. Random distributions conform to a Poisson distribution in which the variance equals the mean. When the variance is less than the mean, the data are distributed more uniformly; when the variance is greater than the mean, the distribution is patchy or aggregated and can be described by a negative binomial distribution. This distribution is determined by two parameters, the mean and the exponent "K" which is a measure of the aggregation of a population. The smaller the value of K, the more aggregated the distribution becomes. Myers (75) compared several indices of dispersion as they relate to biologically signficant events. She found that the ratio of variance to mean was a fairly good estimate of patchiness and was only weakly correlated with population density. The standardized Morisita's coef- ficient (108) and Green's coefficient (43) were both statistically independent of population density and were also highly correlated to the degree of patchiness. If several indices agree, then strong state- ments can be made about dispersal when the relationships between density and dispersal may be changing through time. Knowledge of nematode distribution patterns is necessary for ac- curate assessment of nematode populations. Improper sampling may fail to detect nematode problems or result in costly treatment of large areas when only a small area is infested. Most sampling sites are 32 33 selected based on presence of symptoms in the host crap or on an assump- tion of random nematode population distribution. An intensive sampling program was designed to study nematode distributions in muck soil under field conditions. Methods Soil samples were taken from plot C-17 at the MSU Muck Farm on November 1, 1977, as previously described. Samples were processed by the centrifugal—flotation method and all plant-parasitic nematodes counted. Nematode population densities were not adjusted for extraction efficiency in this experiment. It was felt this would complicate the statistical analysis. The frequency distributions of the three nematode species were calculated for the whole plot and for each of the four subplots. Several theoretical distributions were examined in an attempt to explain the nematode distributions. Data from the November 1977 samples were used to design the layout for a field experiment to determine the effect of host crop on horizontal distribution patterns of nematodes (Appendix B). The plot was divided into six blocks of relatively similar population densities. Carrots, celery or onions were each planted in three-row beds across the block. A fourth treatment consisted of a bed left untreated and in which a natural population of weeds was allowed to develop. Crop beds were kept weed-free by use of commercial herbicides and by handweeding at intervals of one to two weeks. Another factor included in this experiment was the effect of crop density on the distribution and development of nematode pOpulations. Crap density was reduced by thinning plant populations to one-half, 34 two—thirds and normal crop population densities. Treatments were ran- domized in a split plot design. Soil samples were collected April 22 and approximately every three weeks beginning June 7. Plant samples were taken, beginning with the June 27 sampling, until harvest (or to the end of the season for weeds). Data from the field experiment were analyzed for the whole plot and for each crop. Nematode populations analyzed include the plant para— sites M, hapla, Pratylenchus penetrans, Paratylenchus hamatus and the predaceous nematodes Mylonchulus s22., Mononchus s22., Aporcelaimus s22. and Mononchoides s22. Occasional individuals of Criconemoides s22. and Butlerius spp. were counted but too few were available for analysis. Large numbers of Aphelenchus s22., Aphelenchoides spp., Acrobeles s2 ., Monhystera spp., Prismatolaimus spp., Psilenchus spp., Rbabditis spp. and Wilsonema spp. were identified but were not counted. A number of other species were also observed but were not identified. Distribution statistics for the nematode populations analyzed were calculated for each crop and for the whole plot (Appendix C). The variance to mean ratio (sz/X) is included because it is a good indicator of aggregation and is a readily understood statistic. However, it is correlated with the population mean (P = 0.05) and therefore changes in this index over time may involve a statistical artifact. Green's index of dispersion (43) is calculated as ((SZ/X) - l/Zx - 1). This index was not significantly correlated with the population mean and therefore, is a valid estimator of the dispersion of a population as the population mean changes. The variance to mean ratio equals one in a completely random distribution (Poisson) and approaches Xx as the 35 population approaches maximum clumping. Green's index equals zero in random distribution and approaches one at maximum clumping. Sampling was also conducted to determine the vertical distribution of nematodes in soil. Soil samples were collected from three depths; 0—10 cm, 15—25 cm, and 50-60 cm; from nine subplots on April 22 and three subplots on June 27. Results Horizontal Distribution: The variance of M, M§2lg_and E, hamatus population densities was larger than the mean in the whole plot and in subplots A, B, C and D (Table 2.1). The variance of E, 2enetrans population density was larger than the mean in all but one subplot. Although there was considerable variability between subplots and between nematode species, the mean and variance of population density for the whole plot for each species cor— responds approximately to the mean of the four subplots. The negative binomial distribution was calculated for each popula— tion. The "K" value obtained for each distribution was similar among the four subplots for each nematode species but differed from that ob- tained from the whole plot with E, 2enetrans and P, hamatus. g, ene- £2222 had an aggregated distribution in most of the subplots but was dispersed more randomly throughout the whole plot. 3, hamatus popula- tions were highly aggregated in the whole plot but were dispersed more randomly in subplots. M, 22212 showed a similar distribution in sub- plots and the whole plot. 36 TABLE 2.1 Nematode frequency distributions from four subplots and the whole plot sampled November 1, 1977. Chi-square values compare observed with expected frequencies from a negative binomial distribution. Statistic Sample _13. 2enetrans M. ha2la _P_. hamatus Sample A 3.28 5.33 0.75 Mean B 3.28 0.80 3.48 C 0.64 2.40 10.04 D 2.24 7.20 16.08 Whole Plot 3.47 5.46 4.22 Sample A 17.00 63.60 1.20 Variance B 18.50 1.60 11.40 C 0.50 16.20 84.10 D 9.40 136.30 207.80 Whole Plot 13.10 44.10 70.70 "K" A 0.74 0.49 1.15 B 0.70 0.82 1;52 C - 0.42 1.36 D 0.70 0.40 1.35 Whole Plot 1.24 0.77 0.27 Chi-square A 44.60** 97.80** 7.80* B 55.00** 18.90** 66.70** C - 38.40** 65.50** D 26.50** 71.40** 44.40** Whole Plot 15.53n.s. 45.00** 108.70** * Significantly different from negative binomial distribution P = 0.10. ** Significantly different from negative binomial distribution P = 0.005. 37 A chi-square test was applied to examine the goodness-of—fit with the negative binomial distribution (Table 2.1). The chi-square values indicate a poor fit with the negative binomial distribution in most cases. Pielou (83) described distributions with added zeros such that sample units would fall into one of two classes that were not visibly distinguishable: one class that for unknown reasons was uninhabitable and the other where individuals are dispersed as usual. Then the empty units consist of uninhabitable units and units which are habitable but chance to be empty. If this distribution could be applied to the nega- tive-binomial, a much better fit between the expected theoretical dis- tribution and the data observed may result. Changes 12_Di§persion Nematode population densities during the 1978 experiment were low- est at planting and increased through the season at varying rates. Changes in dispersion indices throughout the year were erratic (Appendix C). Green's index showed only slight differences through the year in most nematodes when data from all crops were considered. Mononchoides .222. is an exception as its population fluctuated dramatically and dis- appeared completely during mid-season. Green's index increased rapidly as the population essentially disappeared from increasing proportions of the plots. When Green's index was computed for each host crop independently, essentially similar results were observed. In general, consistent changes over the year were not found. The values were significantly higher for the individual crops than when data for all crops were com- bined. This may be a statistical artifact due to an insufficient 38 number of samples. Green (43) suggested that at least 50 samples were needed for calculation of any coefficient of dispersion, particularly when the variance is high. This phenomenon is apparent in the data for Monochoides 322. in which the maximum clumping resulted when only one sample contained the nematode. In summary, dispersion of plant-parasitic or predaceous nematodes does not change significantly throughout a growing season except in re- sponse to dramatic changes in population density. The influence of the host crop apparently is not overwhelming although more study is needed to conclusively describe this interaction. Vertical Distribution The vertical distribution of nematodes was limited to the upper 0.5 m of soil (Table 2.2). No plant-parasitic nematodes, and few, if any, free living nematodes were found in samples collected from the 50 to 60 cm depth. Slightly fewer plant-parasitic nenatodes were recovered from the 0 to 10 cm depth than from the 15 to 25 cm depth although this difference was not statistically significant. Soil in the upper 10 cm was relatively dry, while at the 50 to 60 cm depth the soil was nearly saturated with water and may have been under anaerobic conditions. 39 TABLE 2.2 Vertical distribution of plant-parasitic nematodes at the MSU Muck Farm. Nematodes/100 cm3 soil Soil (cm) M. 22113 P, 2enetrans _R. hamatus 0-10 31 a1 8 a 16 a 15-25 34 a 32 a 30 a 50—60 0 b 0 a 0 b lColumn means followed by the same letter are not sig- nificantly different (P = 0.05) by the Student-Newman- Keuls Multiple Range Test. PHYSICAL FACTORS The Muck Farm is classified as a Houghton muck. Water levels are managed by tile drainage and sprinkler irrigation as needed. Fertilizer and pesticides are applied under normal commercial vegetable production practices. Given these circumstances, soil temperature and soil fertility factors were monitored to determine their influence on the ontogeny of the host and the development of nematode populations. Methods Soil temperature was monitored continuously using a T603 Three Point Thermograph (Weather Measure Co., Sacramento, California). The three sensing probes are mercury filled, corrosion resistant, stainless steel about 22 cm long and 1 cm in diameter. They were inserted paral- lel to the soil surface and was interrupted only for soil tillage oper- ations or by mechanical failures. The data were used to calculate de~ gree days by the method of Baskerville-Emin with a base of 10 C. Soil samples were taken at mid-season and were analyzed for pH, phosphorous, potassium, calcium and magnesium. Samples were analyzed by the Michigan State University Soil Testing Laboratory using standard procedures. Results Soil Temperatures: Soil temperatures at the 5 cm depth reached a minimum of —3 C in mid-January and were below freezing from December 28, 1977, until 40 41 February 26, 1978. Temperatures remained at 0 C or above at the 15 and 30 cm depths and the soil probably remained unfrozen throughout the winter. Temperatures ranged between 0 and 3 C from mid-December until the snow cover disappeared in early April. Temperatures rose rapidly at the 5 cm depth but high soil moisture prevented a similar increase at lower depths for several weeks. Temperatures were relatively con- stant around 25, 22 and 18 :5 C at the 5, 15 and 30 cm depths, respect- ively, throughout most of the 1978 growing season, but began to drop after mid-September. Early snow cover in November of 1978 resulted in temperatures remaining 1-2 C warmer in December 1978 than in December 1977. Severe cold weather resulted in temperatures during the remainder of the winter were similar to those of the previous year. The 15 cm depth most closely corresponded to the average root con— ditions and further analyses of population dynamics were based on degree day accumulations at this depth. The corresponding calendar dates are given in Table 3.1. The cumulative degree day curve at the 15 cm depth follows that at the 30 cm depth during the early season but closely approximates the 5 cm depth during the latter part of the sea— son (Figure 3.l). This is probably a function of the soil moisture of the three sampling depths. Soil Nutrient Status Soil pH varied from 5.7 to 6.4 (mean 6.07, std. dev. 0.16). the highest pH was in the eastern blocks with the lowest readings in the center two blocks. Although this pattern gives statistically signifi- cant correlation coefficients with the nematode distribution patterns 42 TABLE 3.1 Degree day accumulations on 1978 sampling dates at three soil depths calculated using the Baskerville-Emin method at a 10 C base. DEGREE—DAYS °C DATE 5 cm 15 cm 30 cm 22 APR 27 0 0 7 JUN 268 181 118 27 JUN 454 349 223 18 JUN 692 592 398 12 AUG 988 887 627 29 AUG 1151 1085 782 19 SEP 1314 1284 953 10 OCT 1363 1374 1050 4 NOV 1370 1385 1060 CUHULQTIVE DEGREE DRYS (10 C) 400. 1600. 1000. 1200. 1400. A A A l A .l A L I 800- 600. 43 ' ’ ’ ant" RPR HRY FIGURE 3.1 . , , r . . . JUN JUL nus SEP bcr Nov Ibtc HONTH Cumulative degree days at three soil depths during 1978. 44 of M, ha2la, P, hamatus, and Aporcelaimus spp. (Table 3.2), the narrow range of the differences in pH is unlikely to have more than a coinci- dental influence on nematode distribution. When the data were analyzed by crop, statistically significant cor— relations were made between pH and several nematode species on some crops. However, no obvious influence could be distinguished that could not be accounted for by a coincidental overlap of distributions. The levels of soil nutrients analyzed were distributed more or less randomly over the plot. No differences could be consistently correlated with any soil nutrient. Potassium was negatively correlated with the population density of E. 2enetrans and was correlated with the host crop. Calcium was negatively correlated with M. 2.3213 population den- sities. Although phosphate has been shown to be important in determining mycorrhizal infection and spore production levels (66) the correlations were not statistically significant in this experiment. When the data were analyzed by crop, other correlations were found to be statistically significant, however, none were consistent across any of the parameters that were measured. Further testing will be necessary to determine the influence, if any, of these levels of soil nutrients on nematode popu— lations. 45 TABLE 3.2 Pearson correlation coefficients of soil test results with various biotic parameters in plot C-17 of the MSU Muck Farm. All data are from samples collected 22. August 29, 1978. pH PO4 K Ca Mg ‘3. 2enetrans -.167 .018 -.263 a -.079 -.092 .M..Mg2l§ .280 b —.038 -.083 -.229 a -.006 'P. hamatus .327 b .024 -.113 -.095 .041 Mylonchulus spp. .194 -.090 .054 -.022 .264 a Mononchus spp. .098 —.053 -.010 -.121 -.124 Aporcelaimus spp. .383 c .041 .127 -.224 a .162 Mononchoides spp. -.104 -.146 .330 b .159 .020 Host Crop -.054 -.085 .319 b —.084 .166 Crop Yield .020 .113 .007 .075 .042 Mycorrhizal Infection .097 -.020 -.l92 -.062 -.124 Mycorrhizal Spores .159 -.l38 -.107 -.176 -.152 Mean (x) 6.07 229.9 d 427.6 d 15085 d 1709 d Std. Dev. (3) .16 50.0 d 121.9 d 1261 d 222 d a Significant at P 0.05 by the Student's "t" Test. Significant at P = 0.01 by the Student's "t" Test. 0" Significant at P = 0.001 by the Student's "t" Test. 0 d Mean and standard deviation of nutrient levels measured as lbs/acre. BIOTIC FACTORS INFLUENCING PLANT PARASITIC NEMATODES Plant—parasitic nematodes have significant interactions with a variety of other taxa in agroecosystems. The complexity of community structure may significantly affect both the host crop and the plant- parasitic nematode populations. Experiments at the MSU Muck Farm were used to study the effect of various biotic components interacting with plant-parasitic nematodes. Methods The plot used in the first experiment was described previously and involved sampling three host crops and weeds grown in muck soils. Pop- ulation densities of predaceous and plant-parasitic nematodes were esti- mated from soil samples. Plant samples were weighed to determine shoot and root weight, and one gram samples of feeder roots were processed by the shaker technique (8) to determine root populations of 2, 2enetrans. Root samples were stained (Appendix A) to determine root populations of M, 22212, Soil and root data were combined to provide an estimate of nematode population densities. Weeds were identified using standard taxonomic keys (12, 41, 49). In some cases, roots were broken off of the plant during sampling and could not be positively identified. Only those roots attached to an identifiable stem were included in the anal- ysis. The second experiment was conducted on another plot at the MSU Muck Farm. The interactions of the nematicides aldicarb (Temik) and 46 47 1,3-D + methyl isothiocyanate (Vorlex), and inoculations of M, 33233 eggs were studied to evaluate their influence on natural nematode pop- ulations. The experiment consisted of eight treatments (Table 4.1) involving various combinations of the following factors: A) Vorlex applied at 50 gal/A as a general soil sterilant three weeks before planting, B) eggs of northern root-knot nematode, produced in green- house cultures, injected with a chisel applicator two weeks after plant- ing, and C) Temik applied at 2.0 lbs ai/A at planting. The treatments were replicated four times and consisted of three rows, 15 m long, spaced 54 cm apart planted to onions (cv Downing Yellow Globe). Standard com- mercial procedures for irrigation, fertilization, and pesticide appli- cation were followed. Soil and plant samples for nematode analysis were taken from the outside rows throughout the season. Yield was deter- mined by harvesting the center row of each plot on October 11. Results Influence 33 Weeds 33_Plant-Parasitic and Predaceous Nematodes: Weeds were very prolific and rapidly covered the subplots. Portu- laca oleracea produced a thick.mat early in the season, but by mid-sea- son Amaranthus retroflexus, 3, ggaecizans, Panicum capillare, Digitaria sanguinalis, and occasionally Echinochloa crusgélli had become estab- lished as the dominant species. These plants continued to appear to dominate the subplots even after senescence had occured, but by late fall Stellaria media had invaded and was the only species which had re- tained its leaves. It became widespread and apparently was the primary producer, especially after heavy frosts had occurred. 48 TABLE 4.1 Nematode population densities and VAM spore densities asso— ciated with nematicide treatments in onion production. Plant parasitic nematode population densities represent the sum of six sample periods. Predaceous nematodes and VAM spore densities represent the sum of three sample periods. Plant-parasitic Predaceous VAM Yield Treatment (cwt/A) nematodes nematodes spores per 100 cm per 100 cm per 100 cm Check 174.8 al 19 a 122 a 37 a Temik 183.8 a 27 a 130 a 53 a Eggs 101.6 a 70 a 88 a 37 a Eggs, Temik 122.0 a 49 a 142 a 46 a Vorlex 178.8 a 30 a 35 a 1441 a Vorlex, Temik 98.1 a 4 a 30 a 29 a Vorlex, eggs 102.1 a 115 a 45 a 50 a Vorlex, eggs 75.2 a 9 a 40 a 52 a and Temik 1Column means followed by the same letter are not significantly differ- ent (P = 0.05) by the Student-Newman-Keuls Multiple Range Test. 49 P. penetrans was isolated from all weed species collected (Table 4.2). High population densities were found in roots of Panicum capil— lare, Digitaria sangginalis, Stellaria media, Echinochloa crusgélli, Sonchus oleraceus and Polygonum pennsylvanicum. Although many mid-sea— son samples of Portulaca oleracea had high root pOpulation densities, the average was relatively low. This may reflect the low initial pop- ulation density of 2, 2enetrans during the early season when E, oleracea was most prevalent. A similar situation apparently occurs with M, 33213 associated with g. oleracea. M, 33233 however was not associated with Panicum capillare, Digitaria sanguinalis, Eragrostis hypnoides, Sonchus oleraceus or Polygonum pennsylvanicum. Stellaria media had high popu- lation densities of M, 33213, More research is needed to determine the importance of this weed in the post-harvest population dynamics of M. 33213. It should be noted that many of the fine feeder roots of many of the weeds broke off and were not included in the samples. This prob- ably results in an underestimate of the population density of endopara- sitic nematodes. The individual influences of concomitant weed populations on free living and ectoparasitic nematode population dynamics was difficult to analyze under field conditions. For this reason, the data for all weed samples were combined. Population densities of g, hamatus remained low throughout the year (Figure 4.1). Weeds apparently are not very suit- able hosts for this species. Population densities of E, 2enetrans de- clined from April (0 degree days) until early June (181 degree days), after which they rose steadily for the remainder of the year. 'M,.M32l3 population densities declined through the end of June (349 degree days). 50 o cm 0 00H H poosuumam enuwaw>amwanoe aacmmemM o so 0 an N maumfinusom mamomuoao manoaom q q om om N voucovaou mfiawuoswa.mwdvaaom 0 OH O ooa m mmmuo w>oq mongoamxm mfiumqmmmum q as me on n mmmuu puny Guam maaawwmauu moanuonfinom c c on 00 OH newsman awaom msxoamouuwu manucmuma< N m ON cc OH newsman wumuumoum manufioommm maauamuma< Nm cm mm no NH vmwsxownu mvaB mHHMHkum 0 mm o mm ca mmmumnmuu mHHmcwmwmwm mwumumwmmw o 2: o 2 em 32,3qu 52348 582mm w w no we so mamamusm mmumumao monasuuom News a. afildaa a. use a g .m Baas uoouiw Hom‘moooquoz ONV cowuoomcH madman mama aanou moaomam pmm3 ocu um moump maeamm cm>mm Hw>o pmuomaaou waefimm .ahmm #092 am: .mopoumsm: owufimmumelucmHa no name: pow: N.q mdm : /I’ \\ I X I. ‘1' \l. 4» ‘ 4> o I 1 I . , s I f . , s 1 0. 600. 1000. DEGREE DRYS (898E 10 0) FIGURE 5.6 Influence of associated crop on pOpulation den- sity of Aporcelaimus spp. 59 in initial (Table 4.3) and final (Table 4.4) population densities. .2. penetrans population densities, however, were negatively correlated with final P, hamatus densities. Population densities of Aporcelaimus spp. were positively associated with the concomitant g, hapla,;£, hama- tus and Mylonchulus spp.; however, the initial positive association be- tween gporcelaimus spp. and P, penetrans was reversed at harvest. Whether these interactions are of biological significance or are simply the result of chance distribution could not be determined by this experiment. Influence gf_Chemical Nematicides 22_Nematodes and yycorrhizae Associated With Onions: The results obtained in the second experiment were not definitive. A variety of factors resulted in a very poor stand of onions and corres- pondingly low yields (Table 4.1). Plant growth parameters (root area, bulb volume, leaf area and weight) were highly variable and there were no significant differences among the treatments. Some mechanical damage occurred during the application of nematode eggs. This may have had a detrimental influence on yields. Field observations indicated that Vor— lex applied at 50 gal/A may have reduced plant growth and yields in some plots; however, no consistent differences could be detected. Later experiments indicated that onion (cv Downing Yellow Globe) is resistant to g. m. This may explain why low populations of plant-parasitic nematodes were observed in spite of the inoculations with eggs. Environmental factors also may have been important in pre- venting build-up of significant populations of plant parasitic nematodes. 60 umme zu: .mucmusum was an Hon.o umma :u: .muamvsum msu so Ho.o umma :u: .muamwsum msu kn no.0 m um unmoflmaawfim o m an unmoamfiamfim n m um unmofimaawam m I sws. m msm. ems. coo. ass. was. m HHN. mmuoam z<> 3:. I «8. RN. 8o. 2:. 2o. Sof .mmm 333088: a flu. soof I 08. H2. H2. 9:. 3 SN. mam 833383 3:. RN. o8. I So. 02. .5. a mmm. .mmm 8588: OS. 8o. H3. So. I «3.- 9:. oHor mam $3581: 9:. 02. H2. 02. NSF I a ma. ofif 835.: .M ME. m8. m2. .5. 9:. a 2m. I So. a .m m SN. :5... a «ma. awn. Sor Ear So. I g .M mmuoam .mmm. .mmm. .mmm. ammm IIIIIII..I .I IIIIIIIIII I. z<> 83050952 magmaoouofiw manocosoz msasnocoag msumama .m Mam“ .2 msmuumsma .m .mmuomm Az<>v mufisuuoo%a umasowanMIumasofimm> cam meowam mwoumam: sm>mm wo mmwufimsmw sowumasaoa Hmfiufisfi mo xauuma sofiumamuuoo aomummm m.q m4m NmH.I I HoH.I mNH.I Noo.I ooH.I moH.I moH.I .amm mmwfionuaocoz cos. 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Response 9_f_ Onion Cultivars to _lg. hapla: Significant differences in all growth parameters were observed be- tween noninoculated plantscfl? some varieties. This may reflect differ- ences in adaptations to the environmental conditions in the greenhouse or varietal differences in growth rate. If the environmental conditions in the greenhouse resulted in reduced growth of some varieties, their sensitivity to g. hapla would be reduced and genetic susceptibility to g; hapla_injury would not be detected (Table 5.4). The high resistance of Downing Yellow Globe onions to y, hapla was confirmed in the cultivar trial. Significantly more y} hapla_were re— covered from Krummery Special than from the other varieties. Population densities from Spartan Banner and Spartan Sleeper were higher than from Downing Yellow Globe or MSU 8155 x 826 (Table 5.5) although these dif- ferences were not significant at the P = 0.05 level. Downing Yellow Globe had relatively low root population densities of y} hapla_indicating the presence of mechanisms for resistance. While root population den— sities in MSU 8155 x 835 were somewhat higher, the comparatively low soil population densities and corresponding reductions in most growth parameters indicate that this variety is severely damaged by the nema- tode, thereby preventing increases in the total population. Krummery Special has a somewhat greater growth potential and apparently can pro- vide a better food base allowing rapid growth of nematode populations. This ultimately results in as much, or more, growth reduction as is 77 Ho>mH Amo.o n mv on» um usouommwv haamoaumaumum uos mum umuuma mama mnu hp vo3oaaom momma nasaou .umoH owamm wamauaaz masoxlsmaszIusovsum man %n H w m.a as on m m.m a on m ~.~ a n.a m ~s.a mama: Im. umaowam m s.m as ss a a.s m as m a.s m m.a m ms.a aumau amuumam a s.a m an m s.m m on m ~.~ a o.~ m an.a mama: In. “magma m o.oa as as m a.s a as a m.m a a.~ a ca.~ sumac amaumam m s.aa as as m o.a m an m s.~ m w.a a ~n.a mama; .m. saw a nmam on o.m~ 0 sea on a.sa a ow m a.s as a.s as ~s.m sumac am: a a.s am sm m m.m m an a m.~ a a.a m am.a mama: .mw amaumam u m.am u s~a o m.ma a oaa a o.a a m.m a m~.m aomao aamaasua as m.aa up as cam m.oa m an m a.s m m.m m wa.N mamas .mq macaw as a.sa cam ma am s.s m as m a.m m w.~ m o~.~ aomno soaam» H wsasson as aaas as :3 as as; 3 U3 “mum U3 mwhm U3 053H0> U3 U CNSUMGHH Hag/fl U H50 amaoa amma amwa aoom boom aasa pass .mGOHso so mum>HuH=o w>Hw mo ucoEQOHm>wc was :u3oaw mnu so mama: mammovwoamz mo moamsHmaH q.m mqmmH Amo.o n mv onu um usmuww IMHv %HHmoHumHumum uos mum kuuoH mswm ocu an vaOHH0m momma casHou H a Rsa as. ama a so mama; .m “83% m o m o m o xomso smuumam m as as a: am so mama; .m 353 m o m o m o xomzo cmuumam a is a... saa a... aa sauna .m saw an mmam m o m o m o xomco sz a sass a sma am 8 mama; .aa. amaomam m o m o m o xomso Hosasum m as m R a as mamas .m 208 m o m o m o #0650 3OHHow H wcHazoa uoa pom uoou HHom «Hum: .m. w wool 50 ooH a a unwaummae am>HuH=u H38. mamas: .z m mamas; am .MHmmz mammoonHmz mo sawmswv soHumHsaoa mnu so mam>HuHso soaco m>Hm mo mocmsHmoH m.m m4moz oH aoo oH mom oN oo< NH ooo oH Hoo NN zoo N zoo NN moo oomo .oNoH zH moomo moon moHs ooeoHoooo< «Hooo ozooooHoHoz mo oneooneoHo U xwvcwma< 108 109 sec. one. me. 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